(12) Patent Application Publication (10) Pub. No.: US 2010/01 12041 A1 Ahmad Et Al
US 201001 12041A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/01 12041 A1 Ahmad et al. (43) Pub. Date: May 6, 2010
(54) ENDOXIFEN COMPOSITIONS AND Publication Classification METHODS (51) Int. Cl. (75) Inventors: Ateeq Ahmad, Wadsworth, IL st 4: :08: (US); Shoukath M. Ali, Vernon A69/10 (2006.015 Hills, IL (US); Moghis U. Ahmad, CD7C 2.3/10 (2006.015 Wadsworth, IL (US); Saifuddin A6IP35/00 (2006.015 Sheikh, Waukegan, IL (US): Imran C07C 215/08 (2006.015 Ahmad, Libertyville, IL (US) A69/48 (2006.01) A6IR 9/28 (2006.01) C d Address:
E.S."9 Ave w- e. (52) U.S. Cl...... 424/450; 514/648; 424/484:564/324; 2275 DEMINGWAY, SUITE 310 424/463; 424/474 MIDDLETON, WI 53562 (US) (57) ABSTRACT (73) Assignee: UNA PHARMACEUTICALS The present invention provides compositions containing INC., Libertyville, IL (US) 9 endoxifen, formulations and liposomes of endoxifen, meth s s ods of preparation of such agents and formulations, and use of 21) Appl. No.: 12/S15.261 Such agents and formulations for the treatment of breast can (21) Appl. No 9 cer and other breast diseases and diseases Susceptible to (22) PCT Filed: Nov. 21, 2007 endoxifen. In particular, the compositions of the present e af-l invention include liposomes, complexes, Vesicles, emulsions, micelles and mixed micelles of endoxifen in which the com (86). PCT No.: PCT/US07/8.5443 positions further contain any of a variety of neutral or charged S371 (c)(1) lipids and desirably, cholesterol and cholesterol derivatives, (2), (4) Date: Jan. 8, 2010 sterols, Z- and E-guggulsterones, phospholipids, fatty acids, s a V8 vitamin D, and vitamin E.The present invention also provides O O methods of preparing endoxifen. The present invention pro Related U.S. Application Data vides methods for treating and preventing breast cancer and (60) Provisional application No. 60/860,420, filed on Nov. other breast related diseases by administrating novel formu 21, 2006, provisional application No. 60/860,788, lations or compositions comprising a therapeutically effec filed on Nov. 22, 2006. tive amount of endoxifen.
1. Mg, THF 2O s
2. THF, Reflux (C) Patent Application Publication May 6, 2010 Sheet 1 of 3 US 2010/01 12041 A1
Figure 1 Patent Application Publication May 6, 2010 Sheet 2 of 3 US 2010/01 12041 A1
Figure 2
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() level. (C) -gC OH Patent Application Publication May 6, 2010 Sheet 3 of 3 US 2010/01 12041 A1
Figure 3
N. / C.e.g. C CYP2D6 - C, O SULT1A1 O) (CYP2b6,CYP2C19, CYP2C9, CYP3A) O) OH Tamoxifen (TAM) 4-hydroxyTAM
CYP3A45 (CYP2C9 + other CYP isoforms) N. / ON/ H oU/N C o CYP2D6 C o C e O C SULT1A1 OH N-desmethylTAM Endoxifen US 2010/01 12041 A1 May 6, 2010
ENDOXFEN COMPOSITIONS AND is present in higher concentration (12.4 ng/mL) than 4-OH METHODS tamoxifen (1 ng/mL) in the human plasma. The majority of genes affected by endoxifen are estrogen-regulated genes (8-9). Use of endoxifen e.g., in place of Tamoxifen, avoids 0001. This application claims priority to U.S. Provisional several metabolic steps that rely on CYP2D6. Application Ser. No. 60/860,420, filed Nov. 21, 2006, and 0007. A strong need exists for methods to treat and to 60/860,788, filed Nov. 22, 2006, both of which are incorpo prevent breast diseases without significant adverse systemic rated herein by reference. side effects, particularly in the premenoposal population. In particular, there is a need for breast cancer treatments and FIELD OF THE INVENTION preventatives that having reduced interactive effect with other medications. 0002. The invention relates to the use of endoxifen in the treatment of mammalian diseases. The invention also relates to liposomes and other formulations such as complexes, SUMMARY OF THE INVENTION vesicles, emulsions, micelles and mixed micelles of endox 0008. The present invention provides methods and com ifen, methods of preparation, and uses, e.g., in the treatment positions for the syntheses and use of active agents such as of human and animal breast diseases. The invention in par anticancer agents. The present invention relates to methods ticular relates to compositions comprising endoxifen-lipid and compositions related to the formulations and uses of complexes, methods of preparation, and their use for the endoxifen, particularly in applications related to the treat treatment of breast diseases, in particular benign and malig ment or prevention of cancer. nant breast disease, enhancing disease regression and reduc 0009. In some embodiments, the present invention pro ing risk of patients developing breast cancer. The invention vides a method of treating a disease, comprising, preparing a further relates to methods of preparing endoxifen and use of composition comprising a therapeutically active amount of endoxifen prepared by inventive method in the treatment of endoxifen and administering the composition. In some human and animal diseases. embodiments, the endoxifen is a free base, or is in the form of a salt. In some preferred embodiments, the endoxifen is in the BACKGROUND OF THE INVENTION form of a salt selected from the group of salts consisting of citrate, acetate, formate, oxalate, tartarate, trifluoroacetane, 0003. Every year more than 210,000 women in the United methane Sulfonate, phosphate, Sulfate, chloride, bromide, States develop breast cancer. One in eight women in the US iodide, lactate, and formate. In some embodiments, the will develop breast cancer during their lives. Approximately endoxifen is predominantly in a form selected from the group 70 percent of breast cancers are fueled by estrogen, and many consisting of E-isomer, Z-isomer, and a mixture of E- and are treated with Tamoxifen, a drug designed to block the Z-isomer. effects of estrogen in breast tissue. 0004 Tamoxifen is an anti-estrogenic drug prescribed for 0010. In some embodiments, method comprises preparing long-term, low dose therapy of breast cancer. It has been a complex comprising an anticancer drug and at least one widely used for more than 30 years for the endocrine treat lipid. In some embodiments, the anticancer drug is endoxifen. ment of all stages of hormone receptor-positive breast cancer In some embodiments, the compounds of the invention are (1-2). Tamoxifen has also been approved for the prevention of not complexed with a lipid. In some embodiments, the com breast cancer (3). In women, one of the adverse events asso pound is in the form of a free base or is in the form of a salt. ciated with Tamoxifen is hot flashes. The risk of hot flashes is 0011. In some embodiments, the present invention pro two to three-folds higher among women who take Tamoxifen vides methods of preparing endoxifen, comprising reacting a than it is for those who do not (4). Selective serotonin-re compound of Formula 5 with acid, wherein the compound of uptake inhibitor (SSRI) antidepressants are prescribed to treat formula 5 has the structure: hot flashes. However, some SSRIs, such as paroxetine and fluoxetine, are known to inhibit cytochrome P450(CYP) 2D6 (5), an enzyme that is important for the metabolism of many C drugs, including Tamoxifen. Our understanding of Tamox ifen metabolism and effect has changed clinical practice through the wide spread recognition that the co-prescription of drugs that inhibit CYP2D6 may compromise Tamoxifen efficacy. It is known that co-administration of paroxetine decreases the plasma concentration of an active metabolite of Tamoxifen, 4-hydroxy-N-desmethyl-Tamoxifen (endox OH ifen). 0005 Endoxifen is generated via CYP3A4-mediated N-demethylation and CYP2D6 mediated hydroxylation of Tamoxifen (see, e.g., FIG. 3). Any drug that can be substrate of CYP3A4 or CYP2D6, especially CYP2D6 e.g., SSRIs, can O decrease the level of endoxifen (6) and thus reduce the thera peutic benefits of Tamoxifen. Therefore, to avoid such drug drug interactions, one should not give them together. 0006 Recently, endoxifen has been shown to be anti-es trogenic in breast cancer cells and to be more potent than Tamoxifen (7). In patients treated with Tamoxifen, endoxifen US 2010/01 12041 A1 May 6, 2010
0012 and, after the reaction of the compound of Formula least one lipid. In preferred embodiments, the at least one 5 with acid, reacting the compound with methylamine. In lipid is selected from the group consisting of egg phosphati Some embodiments, the compound of Formula 5 is prepared dylcholine (EPC), egg phosphatidylglycerol (EPG), soy by reacting compound of formula 4 phosphatidylcholine (SPC), hydrogenated soy phosphatidyl choline (HSPC), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmi toylphosohatidylcholine (DPPC), disteroylphosphatidylg lycerol (DSPG), dipalmitoylphosphatidylglycerol (DMPG), cholesterol (Chol), cholesterol sulfate and its salts (CS), cho lesterol hemisuccinate and its salts (Chems), cholesterol phosphate and its salts (CP), cholesterylphosphocholine and other hydroxycholesterol or amino cholesterol derivatives, cholesteryl Succinate, cholesteryl oleate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG), coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone, and calciferol, E-guggulsterone, Z-guggul ac sterone, mixture of E- and Z-guggulsterone, monoglycerides, with a compound of Formula 3. diglycerides, triglycerides, carbohydrate-based lipids selected from a group consisting of galactolipid, mannolipid, galactolecithin, B-sitosterol, Stigmasterol, Stigmastanol, lanosterol, C.-spinasterol, lathosterol, campesterol, phos Br phatidylcholine, phosphatidylglycerol, phosphatidylethano lamine, phosphatidylserine, phosphatdylinositol, phospha tidic acid, and pegylated derivatives of distearoylphosphatidylglycerol, dipalmitoylphosphatidylg lycerol, dimyristoylphosphatidylglycerol, and dioleoylphos phatidylglycerol. O O 0016. In some embodiments, a composition according to the present invention comprises endoxifen, cholesterol and/or cholesterol derivatives, and one or more phospholipids. In Some preferred embodiments, the composition comprises a cholesterol derivative, and the cholesterol derivative is cho 0013. In some embodiments, the compound of Formula 3 lesteryl sulfate. In some embodiments, at least one of the is prepared by reacting compound of Formula 1 phospholipids is hydrogenated soy phosphatidylcholine or soy phosphatidylcholine. 0017. In some embodiments of the methods and compo sitions of the present invention, the composition comprises a form selected from the group consisting of powder, solution, emulsion, micelle, liposome, lipidic particle, gel, and paste form. In some preferred embodiments, the preparing of the composition comprising a complex comprises preparing said complex in a lyophilized form. In some embodiments, the OH preparing the complex in a lyophilized form comprises using a cryoprotectant, wherein said cryoprotectant comprises one with a compound of Formula 2. or more Sugars selected from the group consisting of treha lose, maltose, lactose, Sucrose, glucose, and dextran. In some embodiments, the composition comprises a tablet or a filled capsule, wherein said tablet or filled capsule optionally com prises an enteric coating material. 0018. In some embodiments of the treatment methods of O the present invention, the disease is caused by cancer or by cancer-causing agents, while in some embodiments, the dis 0014. In some embodiments, the present invention pro ease is benign breast disease. vides methods of purifying the endoxifen as described above, 0019. In some embodiments, the administering comprises comprising crystallizing the endoxifen and/or chromato oral, intravenous, Subcutaneous, percutaneous, parenteral, graphically treating said endoxifen to produce a purified intraperitoneal, rectal, vaginal, and/or topical delivery said preparation of endoxifen, wherein the purified preparation of composition to said Subject. endoxifen contains predominantly E-isomer, predominantly 0020. In some embodiments, the composition comprises a Z-isomer, or mixture of E- and Z-isomers of endoxifen. penetration enhancer, wherein said penetration enhancer 0.015. As described above, in some embodiments, the comprises at least one saturated or unsaturated fatty acid invention provides endoxifen preparations comprising at ester. US 2010/01 12041 A1 May 6, 2010
0021. In some embodiments, the composition comprising containing a penetration enhancer, an aqueous vehicle, an endoxifen is formulated in a hydroalcoholic gel, a hydroal alcoholic vehicle and a gelling agent. coholic Solution, a patch, a cream, an emulsion, a lotion, an 0037. In some embodiments, the hydroalcoholic compo ointment, a powder or an oil. sition comprises a neutralizing agent. 0022. In some embodiments, the composition comprising 0038. In some embodiments, the hydroalcoholic compo endoxifen is formulated in a hydroalcoholic composition sition comprises endoxifen at about 0.01% to 0.20% by containing a penetration enhancer, an aqueous vehicle, an weight; isopropyl myristate at about 0.1% to 2.0%, preferably alcoholic vehicle and a gelling agent. 0.5% to 2.0% by weight: alcohol at about 50.0% to 80.0%, 0023. In some embodiments, the hydroalcoholic compo preferably about 60.0% to 75.0% by weight; aqueous vehicle sition comprises a neutralizing agent. at about 20.0% to 60.0%, preferably 25.0% to 50.0% by 0024. In some embodiments, the hydroalcoholic compo weight; and gelling agent at about 1.0% to 10.0%, preferably sition comprises endoxifen at about 0.01% to 0.20% by about 0.5% to 5.0% by weight. In some embodiments, the weight; isopropyl myristate at about 0.1% to 2.0%, preferably wherein the percentage of components is weight to weight of 0.5% to 2.0% by weight; alcohol at about 50.0% to 80.0%, the composition. preferably about 60.0% to 75.0% by weight; aqueous vehicle 0039. The present invention is not limited to the compo at about 20.0% to 60.0%, preferably 25.0% to 50.0% by sitions and methods described above. It should be understood weight; and gelling agent at about 1.0% to 10.0%, preferably that variations of the above compositions and methods about 0.5% to 5.0% by weight. In some embodiments, the described elsewhere herein or that are understood by a skilled wherein the percentage of components is weight to weight of artisan in view of the present disclosure are included within the composition. the invention. 0025. In some embodiments, the alcohol is ethanol or isopropanol, and constitutes in absolute form. DESCRIPTION OF THE DRAWINGS 0026. In some embodiments, the aqueous vehicle is a phosphate buffered solution. 0040 FIG. 1 diagrams compound I. 0027. In some embodiments, the gelling agent is selected 0041 FIG. 2 diagrams embodiments for synthesis of com from the group consisting of polyacrylic acid, hydroxypro pounds 3, 5, and I. pylcellulose and a cellulose derivative other than hydroxypro 0042 FIG. 3 diagrams portions of Tamoxifen metabolic pylcellulose. pathways. 0028. In some embodiments, the hydroalcoholic compo sition further comprises a neutralizing agent, wherein said DEFINITIONS neutralizing agent is selected from the group consisting of 0043. As used herein, the terms “host,” “subject' and Sodium hydroxide, potassium hydroxide, ammonium “patient” refer to any animal, including but not limited to, hydroxide, aminomethylpropanol, arginine, trolamine, and human and non-human animals (e.g., dogs, cats, cows, tromethamine, and wherein said neutralizing agent exists at a horses, sheep, poultry, fish, crustaceans, etc.) that is studied, neutralizing agent/gelling agent ratio of about 1:1 to about analyzed, tested, diagnosed or treated. As used herein, the 4:1. terms “host,” “subject' and “patient” are used interchange 0029. In some embodiments, the invention provides meth ably, unless indicated otherwise. ods of delivering endoxifen, comprising: providing any of the 0044 As used herein, the terms “subject at risk of cancer' above described compositions and delivering the composi refers to a subject identified as being at risk for developing tion so as to expose the composition to a cell. cancer, e.g., by prior health history, genetic data, etc. 0030. In some embodiments, the cell is in vivo. 0045. As used herein, the term “anticancer drug” refers to 0031. In some embodiments of the invention, the host is a an agent used to treat or prevent cancer. Such agents include, mammal. but are not limited to, Small molecules, drugs, antibodies, 0032. The present invention also provides methods of pharmaceuticals, and the like. inhibiting hormone-dependent breast carcinoma in a mam 0046. As used herein, the term “effective amount” refers to mal comprising administering any of the above compositions the amount of an active composition (e.g., a pharmaceutical to the mammal. compound or composition provided as a component in a lipid 0033. The present invention further provides methods of formulation) sufficient to effect beneficial or desired results. inhibiting a cancer in a mammal, said cancer including, but An effective amount can be administered in one or more not limited to, lung cancer, colon cancer, breast cancer, leu administrations, applications or dosages and is not intended kemia, renal cancer, melanoma, cancer or the central nervous to be limited to a particular formulation or administration system, and prostate cancer in a mammal; the method com rOute. prising administering any of the above compositions to said 0047. As used herein, the terms “active' or “pharmaceu mammal (e.g., a human). tically active' as used in reference to an agent, drug, compo 0034. The present invention further provides composi sition, or compound, refers to an agent that, upon administra tions comprising a therapeutically active amount of a com tion or application, causes a beneficial, desired, or expected plex comprising endoxifen and at least one lipid, wherein said result. The administration may be in one or more administra endoxifen is a free base or is in the form of a salt. tions, applications or dosages and is not intended to be limited 0035. In some embodiments, the composition comprising to a particular formulation or administration route. The term endoxifen is formulated in a hydroalcoholic gel, a hydroal is not limited to any particular level of activity. coholic Solution, a patch, a cream, an emulsion, a lotion, an 0048. The terms “agent” and “compound are used herein ointment, a powder or an oil. interchangeably to refer to any atom, molecule, mixture, or 0036. In some embodiments, the composition comprising more complex composition having an attributed feature. For endoxifen is formulated in a hydroalcoholic composition example, an “active agent” or “active compound” refers to US 2010/01 12041 A1 May 6, 2010
any atom, molecule, preparation, mixture, etc., that, upon tration may be in one or more administrations, applications or administration or application, causes a beneficial, desired, or dosages, and is not intended to be limited to a particular expected result. administration route. 0049. As used herein, the term “treating includes admin 0055 As used herein, the term “co-administration” refers istering therapy to prevent, cure, or alleviate/prevent the to the administration of at least two agent(s) (e.g., two sepa rate lipid compositions, containing different active com symptoms associated with, a specific disorder, disease, injury pounds) or therapies to a Subject. In some embodiments, the or condition. co-administration of two or more agents or therapies is con 0050. As used herein, the term “treatment' or grammatical current. In other embodiments, a first agent/therapy is admin equivalents encompasses the improvement and/or reversal of istered prior to a second agent/therapy. Those of skill in the art the symptoms of disease (e.g., cancer), or reduction of risk of understand that the formulations and/or routes of administra occurrence of disease. A compound which causes an tion of the various agents or therapies used may vary. The improvement in any parameter associated with disease when appropriate dosage for co-administration can be readily used in the screening methods of the instant invention may determined by one skilled in the art. In some embodiments, thereby be identified as a therapeutic compound. The term when agents or therapies are co-administered, the respective “treatment” refers to both therapeutic treatment and prophy agents or therapies are administered at lower dosages than lactic or preventative measures. For example, those who may appropriate for their administration alone. Thus, co-adminis benefit from treatment with compositions and methods of the tration is especially desirable in embodiments where the co present invention include those already with a disease and/or administration of the agents or therapies lowers the requisite disorder (e.g., cancer, or symptoms or pathologies consistent dosage of a potentially harmful (e.g., toxic) agent(s). with cancer) as well as those in which a disease and/or disor 0056. As used herein, the term “toxic' refers to any detri der is to be prevented (e.g., using a prophylactic treatment of mental or harmful effects on a Subject, a cell, or a tissue as compared to the same cell or tissue prior to the administration the present invention). of the toxicant. 0051. As used herein, the term “at risk for disease' refers 0057. As used herein, the term “pharmaceutically puri to a subject (e.g., a human) that is predisposed to experiencing fied’ refers to a composition of sufficient purity or quality of a particular disease. This predisposition may be genetic (e.g., preparation for pharmaceutical use. a particular genetic tendency to experience the disease. Such 0058 As used herein, the term “purified” refers to a treat as heritable disorders), or due to other factors (e.g., age, ment of a starting composition to remove at least one other weight, environmental conditions, exposures to detrimental component (e.g., another component from a starting compo compounds present in the environment, etc.). Thus, it is not sition (e.g., plant or animal tissue, an environmental sample intended that the present invention be limited to any particular etc.), a contaminant, a synthesis precursor, or a byproduct, risk, nor is it intended that the present invention be limited to etc.), such that the ratio of the purified component to the any particular disease. removed component is greater than in the starting composi 0052. As used herein, the term “suffering from disease' tion. refers to a subject (e.g., a human) that is experiencing a 0059. As used herein, the term “pharmaceutical composi particular disease. It is not intended that the present invention tion” refers to the combination of an active agent (e.g., an be limited to any particular signs or symptoms, nor disease. active pharmaceutical compound) with a carrier, inert or Thus, it is intended that the present invention encompasses active (e.g., a phospholipid), making the composition espe Subjects that are experiencing any range of disease (e.g., from cially Suitable for diagnostic or therapeutic use in vitro, in sub-clinical manifestation to full-blown disease) wherein the vivo or ex vivo. Subject exhibits at least some of the indicia (e.g., signs and 0060. The terms “pharmaceutically acceptable' or “phar symptoms) associated with the particular disease. macologically acceptable, as used herein, refer to composi 0053 As used herein, the terms “disease' and “pathologi tions that do not substantially produce adverse reactions, e.g., cal condition' are used interchangeably to describe a state, toxic, allergic, or immunological reactions, when adminis signs, and/or symptoms that are associated with any impair tered to a subject. ment of the normal state of a living animal or of any of its 0061. As used herein, the term “topically’ refers to appli organs or tissues that interrupts or modifies the performance cation of the compositions of the present invention to the of normal functions, and may be a response to environmental Surface of the skin and mucosal cells and tissues (e.g., alveo factors (such as emotional trauma, physical trauma, malnu lar, buccal, lingual, masticatory, or nasal mucosa, and other trition, industrial hazards, or climate), to specific infective tissues and cells which line hollow organs or body cavities). agents (such as worms, bacteria, or viruses), to inherent 0062. As used herein, the term “pharmaceutically accept defect of the organism (such as various genetic anomalies, or able carrier refers to any of the standard pharmaceutical to combinations of these and other factors. carriers including, but not limited to, phosphate buffered 0054 As used herein, the term “administration” refers to saline solution, water, emulsions (e.g., Such as an oil/water or the act of giving a drug, prodrug, or other active agent, or water/oil emulsions), and various types of wetting agents, any therapeutic treatment (e.g., compositions of the present and all solvents, dispersion media, coatings, sodium lauryl invention) to a physiological system (e.g., a Subjector in vivo, Sulfate, isotonic and absorption delaying agents, disintigrants in vitro, or ex vivo cells, tissues, and organs). Exemplary (e.g., potato starch or Sodium starch glycolate), and the like. routes of administration to the human body can be through the The compositions also can include Stabilizers and preserva eyes (ophthalmic), mouth (oral), skin (transdermal), nose tives. For examples of carriers, stabilizers, and adjuvants. (nasal), lungs (inhalant), rectal, vaginal, oral mucosa (buc (See e.g., Martin, Remington's Pharmaceutical Sciences, cal), ear, by injection (e.g., intravenously, Subcutaneously, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated intratumorally, intraperitoneally, etc.) and the like. Adminis herein by reference). Moreover, in certain embodiments, the US 2010/01 12041 A1 May 6, 2010
compositions of the present invention may be formulated for 0069. As used herein, the term “dried” as used in reference horticultural or agricultural use. Such formulations include to a composition refers to removing the solvent component or dips, sprays, seed dressings, stem injections, sprays, and components to levels that no longer Support chemical reac mists. tions. The term is also used in reference to a composition that has been dried (e.g., a dried preparation or dried composi 0063 As used herein, the term “pharmaceutically accept tion). Those of skill in the art will appreciate that a composi able salt” refers to any salt (e.g., obtained by reaction with an tion may be “dried while still having residual solvent or acid or a base) of a compound of the present invention that is moisture content after, e.g., lyophilization, or that a dried physiologically tolerated in the target Subject (e.g., a mam composition may, after the end of a drying process, absorb malian Subject, and/or in Vivo or ex vivo, cells, tissues, or moisture hygroscopically, e.g., from the atmosphere. The organs). “Salts' of the compounds of the present invention term “dried encompasses a composition with increased may be derived from inorganic or organic acids and bases. moisture content due to hygroscopic absorption. Examples of acids include, but are not limited to, hydrochlo ric, hydrobromic, Sulfuric, nitric, perchloric, fumaric, maleic, 0070. As used herein, the term “protective agent” refers to phosphoric, glycolic, lactic, Salicylic, succinic, toluene-p- a composition or compound that protects the activity or integ rity of an active agent (e.g., an anticancer drug) when the Sulfonic, tartaric, acetic, citric, methanesulfonic, ethane active agent is exposed to certain conditions (e.g., drying, Sulfonic, formic, benzoic, malonic, Sulfonic, naphthalene-2- freezing). In some embodiments, a protective agent protects sulfonic, benzenesulfonic acid, and the like. Other acids, such an active agent during a freezing process (i.e., it is a "cryo as oxalic, while not in themselves pharmaceutically accept protectant”). Examples of protective agents include but are able, may be employed in the preparation of salts useful as not limited to non-fat milk Solids, trehalose, glycerol, betaine, intermediates in obtaining the compounds of the invention Sucrose, glucose, lactose, dextran, polyethylene glycol, Sor and their pharmaceutically acceptable acid addition salts. bitol, mannitol, poly vinyl propylene, potassium glutamate, 0064. Examples of bases include, but are not limited to, monosodium glutamate, Tween 20 detergent, Tween 80 deter alkali metal (e.g., Sodium) hydroxides, alkaline earth metal gent, and an amino acid hydrochloride. (e.g., magnesium) hydroxides, ammonia, and compounds of 0071. As used herein, the term “excipient” refers to an formula NW.", wherein W is C alkyl, and the like. inactive ingredient (i.e., not pharmaceutically active) added 0065. Examples of salts include, but are not limited to: to a preparation of an active ingredient. The gelling and acetate, adipate, alginate, aspartate, benzoate, benzene protective agents described herein may be referred to gener Sulfonate, bisulfate, butyrate, citrate, camphorate, camphor ally as “excipients.” Sulfonate, cyclopentanepropionate, digluconate, dodecylsul (0072. As used herein, the term “kit” refers to any delivery fate, ethanesulfonate, fumarate, flucoheptanoate, system for delivering materials. In the context of kinase activ glycerophosphate, hemisulfate, heptanoate, hexanoate, chlo ity or inhibition assays, such delivery systems include sys ride, bromide, iodide, 2-hydroxyethanesulfonate, lactate, tems that allow for the storage, transport, or delivery of reac maleate, methanesulfonate, 2-naphthalenesulfonate, nicoti tion reagents and/or Supporting materials (e.g., buffers, nate, oxalate, palmoate, pectinate, persulfate, phenylpropi written instructions for performing the assay etc.) from one onate, picrate, pivalate, propionate, Succinate, tartrate, thio location to another. For example, kits include one or more cyanate, tosylate, undecanoate, and the like. Other examples enclosures (e.g., boxes) containing the relevant reaction of salts include anions of the compounds of the present inven reagents and/or Supporting materials. As used herein, the term tion compounded with a suitable cation such as Na', NH.", “fragmented kit' refers to delivery systems comprising two or and NW (wherein W is a C alkyl group), and the like. For more separate containers that each contains a subportion of therapeutic use, salts of the compounds of the present inven the total kit components. The containers may be delivered to tion are contemplated as being pharmaceutically acceptable. the intended recipient together or separately. For example, a However, salts of acids and bases that are non-pharmaceuti first container may contain an agent for use in an assay, while cally acceptable may also find use, for example, in the prepa a second container contains standards for comparison to test ration or purification of a pharmaceutically acceptable com compounds. The term “fragmented kit' is intended to encom pound. pass kits containing Analyte Specific Reagents (ASR's) regu 0066 For therapeutic use, salts of the compounds of the lated under section 520(e) of the Federal Food, Drug, and present invention are contemplated as being pharmaceuti Cosmetic Act, but are not limited thereto. Indeed, any deliv cally acceptable. However, salts of acids and bases that are ery system comprising two or more separate containers that non-pharmaceutically acceptable may also find use, for each contains a subportion of the total kit components are example, in the preparation or purification of a pharmaceuti included in the term “fragmented kit.” In contrast, a “com cally acceptable compound. bined kit' refers to a delivery system containing all of the 0067. As used herein, the term “hydroalcoholic” as used in components of a reaction assay in a single container (e.g., in reference to a substance or composition indicates that said a single box housing each of the desired components). The Substance or composition comprises both water and alcohol. term "kit' includes both fragmented and combined kits. 0068. As used herein, the term “gelling agent” refers to a composition that, when dissolved, Suspended or dispersed in DETAILED DESCRIPTION OF THE INVENTION a fluid (e.g., an aqueous fluid Such as water or a buffer solu tion), forms a gelatinous semi-solid (e.g., a lubricant gel). 0073. The present invention provides compositions and Examples of gelling agents include but are not limited to methods for delivering endoxifen of Formula I, e.g., to a hydroxyethyl cellulose, hydroxymethyl cellulose, hydrox mammalian host. In some embodiments of the present inven ypropyl guar, methyl cellulose, ethyl cellulose, hydroxypro tion endoxifen is an E-isomer, while in other embodiments, it pyl cellulose, Sodium carboxymethyl cellulose, carbomer, is a Z-isomer, while it is still in other embodiments, it is a alginate, gelatin, and poloxamer. mixture of E- and Z-isomers US 2010/01 12041 A1 May 6, 2010
Solvent, or as mixture of Solvents such as hexane-ethyl acetate, chloroform-acetone, chloroform-methanol, dichlo romethane-methanol, and the like. When a mixture of two CH3 Solvents is used in the present invention, examples of ratios of N one solvent to another are e.g., in a range such as 9:1 to 1:9. o-U/ \V (e.g., 8:2, 7:3: 6:4; 5:5; 4:6; 3:7: 2:8; 1:9, and the like.) However, mixtures for use in the present invention are not limited to these ratios, or to mixtures comprising only two Solvents. C. C. 0078 Solvents that find use in the preparation of endox ifen according to the present invention include but are not limited to tetrahydrofuran, dichloromethane, chloroform, H3CHC O 1,2-dichloroethane, acetonitrile, N,N'-dimethylformamide, dimethylsulfoxide, toluene, pyridine, methanol, ethanol, iso OH propanol, acetone, 2-butanone, hexane, heptane, pentane, ethyl acetate, and the like. 0074 An example of the present invention includes 0079 Acids that find use in the preparation of endoxifen endoxifen, analogues of endoxifen, and derivatives of endox according to the present invention include, but are not limited ifen, including but not limited to endoxifen, Tamoxifen, and to, Sulfuric acid, hydrochloric acid, acetic acid, trifluoroacetic 4-hydroxytamoxifen. The present invention also find use with acid, phosphoric acid, p-toluenesulfonic acid, methane otherantineoplastic agents such as paclitaxel, docetaxel, mel Sulfonic acid, nitric acid, and the like. phalan, chlormethine, extramustinephosphate, uramustine, 0080 Intermediates and final products of the present ifosfamide, mannomustine, trifosfamide, Streptozotocin, invention can be purified by column chromatography using a mitobronitol, mitoxantrone, methotrexate, fluorouracil, cyt single or a mixture of common organic solvents such as arabine, tegafur, idoxide, taxol, paclitaxel, daunomycin, hexane pentane, heptane, ethyl acetate, methylene chloride, daunorubicin, bleomycin, amphotericin, carboplatin, cispl chloroform, methanol, acetone, and the like. atin, BCNU, Vincristine, camptothecin, SN-38, doxorubicin, I0081. As noted above, intermediates and final product of and etopside. Also included are steroidal and non-steroidal the present invention, may, in some embodiments, be purified inhibitors used in cancer treatment, Such as bicautamide, by crystallization. Solvents that find use in the crystallization exemestane, formestane, letrozole, anastrazole and their ana of intermediates and products include but are not limited to logues. hydrocarbons such as pentanes, hexanes, heptanes, and the 0075 Endoxifen of Formula I can be prepared by any like, benzene; toluene; ethyl acetate; acetonitrile; chlorinat desired method for use in the treatments of the present inven ing solvents such as methylene chloride, chloroform, 1.2- tion but, in Some embodiments, the present invention pro dichloromethane, and the like, ketones, for example, acetone, vides particular methods for the preparation of endoxifen. 2-butanone, and the like; ethers such as diethyl ether, diiso One preferred method of the present invention is set forth in propyl ether, methyl butyl ether, tetrahydrofuran; alcohols FIG. 2. In this method, 4-bromophenol 1 is reacted with Such as methanol, ethyl alcohol, isopropyl alcohol, and the 3,4-dihydropyran 2 in the present of acid (e.g., Sulfuric acid like. A solvent for crystallization can be used as a single and the like), to give compound 3. Compound 3 is then Solvent or mixture of solvents. Exemplary mixtures include, reacted with magnesium turning in a Suitable anhydrous sol e.g., hexane-ethyl acetate, chloroform-acetone, chloroform vent (e.g., tetrahydrofuran and the like). This is followed by methanol, dichloromethane-methanol, and the like. When a reaction with 1-4-(2-chloroethoxy)phenyl)-2-phenyl-1-bu mixture of two solvents is used in the present invention, tanone 4 to provide compound 5 which, on dehydration/ examples of ratios of one solvent to another are e.g., in a range deprotection in presence of acid in a Suitable solvent (e.g., such as 9:1 to 1:9, (e.g., 8:2, 7:3: 6:4;5:5; 4:6; 3:7: 2:8; 1:9, methanol and the like), produces compound 6. Reaction of and the like.) However, mixtures for use in the present inven yielded compound 6 with methylamine in a suitable solvent tion are not limited to these ratios, or to mixtures comprising (e.g., isopropanol and the like) provides endoxifen I. only two solvents. 0076. In some embodiments of the present invention, a I0082 One object of the present invention is to provide mixture of E- and Z-isomers of endoxifen can be separated to E-endoxifen or Z-endoxifen with at least 80% purity, such as provide the purified preparations of E- and Z-isomer of at least 90% pure or at least 95% pure or at least 98% pure or endoxifen. The separation of E- and Z-isomers of endoxifen at least 99% pure or at least 100% pure. in the present invention can be done, e.g., by crystallization, I0083. Another object of the present invention is to provide or purification by liquid column chromatography (LC), or solubilized endoxifenin, e.g., aqueous acid. Suitable acids for high pressure liquid column chromatography (HPLC). solubilizing endoxifen include but are not limited to formic 0077 Suitable solvents that can be employed in present acid, acetic acid, propionic acid, butyric acid, trifluoroacetic invention for the separation of E- and Z-isomers of endoxifen acid, lactic acid, tartaric acid, oxalic acid, malonic acid, Suc include but are not limited to hexanes, heptanes, and the like, cinic acid, and the like. The pH of the acidic solution com benzene: toluene; ethyl acetate; acetonitrile; chlorinating Sol prising endoxifen can be adjusted with suitable base or buff vents such as methylene chloride, chloroform, 1,2-dichlo ers. Examples of base and buffers include but are not limited romethane, and the like, ketones, (e.g., acetone, 2-butanone, to Sodium hydroxide, Sodium acetate, sodium lactate, sodium and the like), ethers such as diethyl ether, diisopropyl ether, Succinate, Sodium monophosphate, Sodium diphosphate, methyl butyl ether, and tetrahydrofuran, alcohols such as Sodium triphosphate, sodium oxalate, sodium tartarate, methanol, ethyl alcohol, and isopropyl alcohol, and the like, ammonium hydroxide, ammonium acetate, and the like. In and water. A solvent for crystallization can be used as a single Some embodiments, a co-solvent can also be used to solubi US 2010/01 12041 A1 May 6, 2010
lize endoxifen. Examples of co-solvent include but are not O), tricosanoic acid (Cso), tetracosanoic acid (C2ao), 10-un limited to ethanol, isopropanol, detergents such as Tween 20 decenoic acid (C), 11-dodecenoic acid (C), 12-tride and Polysorbate, and the like cenoic acid (C1-), myristoleic acid (C1). 0084. In certain preferred embodiments, the pH of a com 10-pentadecenoic acid (Cs), palmitoleic acid (C), oleic position containing endoxifen according to the present inven acid (Cs), linoleic acid (Cs), linolenic acid (Cs), tion are between about 4.0 and about 8.0, and preferably eicosenoic acid (Co.), eicosdienoic acid (Co.), eicosa between about 5.0 and about 8.0, and most preferably trienoic acid (Co.), arachidonic acid (cis-5,8,11,14-eico between about 5.5 and about 7.5. satetraenoic acid), and cis-5,8,11,14, 17-eicosapentaenoic 0085. In some embodiments, the present invention relates acid, among others. Other fatty acids also can be employed in to compositions and methods for delivery of endoxifen or the compositions. Examples of Such include Saturated fatty endoxifen-lipid complexes to a mammalian host. Any Suit acids such as ethanoic (or acetic) acid, propanoic (or propi able amount of endoxifen can be used in complex formation. Suitable amounts of endoxifen are those amounts that can be onic) acid, butanoic (or butyric) acid, hexacosanoic (or stably incorporated into the complexes of the present inven cerotic) acid, octacosanoic (or montanic) acid, triacontanoic (or melissic) acid, dotriacontanoic (or lacceroic) acid, tetra tion. triacontanoic (or gheddic) acid, pentatriacontanoic (or cero I0086. In some embodiments, the inventive composition plastic) acid, and the like; monoethenoic unsaturated fatty comprises a lipid complex with endoxifen in which the com acids such as trans-2-butenoic (or crotonic) acid, cis-2- plex desirably contains lipid or a mixture of lipids. Com butenoic (or isocrotonoic) acid, 2-hexenoic (or isohydrosor plexes can be in the form, e.g., of micelles, Vesicles or emul bic) acid, 4-decanoic (or obtusilic) acid, 9-decanoic (or sions without exclusion of other forms. The micelles of the caproleic) acid, 4-dodecenoic (or linderic) acid, 5-dode present invention can be in the form of monomeric, dimeric, cenoic (or denticetic) acid, 9-dodecenoic (or lauroleic) acid, polymeric or mixed micelles. The complexes including 4-tetradecenoic (or tsuZuic) acid, 5-tetradecenoic (or physe micelles and emulsions are predominately in the size range of teric) acid, 6-octadecenoic (or petroselenic) acid, trans-9- 50 nm-20 micron, preferably in size range of 50 nm-5 micron. octadecenoic (or elaidic) acid, trans-11-octadecenoic (or vac In the complexes, the active agent can be bound to the lipid by cinic) acid, 9-eicosenoic (orgadoleic) acid, 11-eicosenoic (or covalent, hydrophobic, electrostatic, hydrogen, or other gondoic) acid, 11-docosenoic (or cetoleic) acid, 13-decose bonds, and is considered bound even where the drug is simply noic (or erucic) acid, 15-tetracosenoic (or nervonic) acid, entrapped within the interior of lipid structures. 17-hexacosenoic (or Ximenic) acid, 21-triacontenoic (or 0087 Endoxifen-lipid complexes may contain e.g., cho lumequeic) acid, and the like; dienoic unsaturated fatty acids lesterols or cholesterol derivatives or a mixture of cholesterol Such as 2.4-pentadienoic (or B-vinylacrylic) acid, 2.4-hexa and cholesterol derivatives. Cholesterol derivatives that find dienoic (or Sorbic) acid, 2,4-decadienoic (or stillingic) acid, use in the present invention include cholesteryl hemisucci 2,4-dodecadienoic acid, 9,12-hexadecadienoic acid, cis-9, nate, cholesteryl Succinate, cholesteryl oleate, cholesteryl cis-12-octadecadienoic (or O-linoleic) acid, trans-9, trans-12 linoleate, cholesteryl eicosapentenoate, cholesteryl lino octadecadienoic (or linlolelaidic) acid, trans-10, trans-12-oc lenate, cholesteryl arachidonate, cholesteryl palmitate, cho tadecadienoic acid, 11,14-eicosadienoic acid, 13,16-docosa lesteryl Stearate, cholesteryl myristate, polyethylene glycol dienoic acid, 17.20-hexacosadienoic acid and the like; derivatives of cholesterol (cholesterol-PEG), water soluble trienoic unsaturated fatty acids such as 6,10,14-hexadecatri cholesterol (for example, cholesterol methyl-f-cyclodex enoic (or hiragonic) acid, 7,10,13-hexadecatrienoic acid, cis trin), coprostanol, cholestanol, or cholestane, cholic acid, 6, cis-9-cis-12-octadecatrienoic (or Y-linoleic) acid, trans-8. cortisol, corticosterone or hydrocortisone and 7-dehydrocho trans-10-trans-12-octadecatrienoic (or B-calendic) acid, cis lesterol. 8,trans-10-cis-12-octadecatrienoic acid, cis-9,cis-12-cis-15 0088. In some preferred embodiments, the compositions octadecatrienoic (or O-linolenic) acid, trans-9, trans-12-trans also include Cl-, 3-, y-tocopherols, vitamin E, calciferol, 15-octadecatrienoic (or O-linolenelaidic) acid, cis-9, trans organic acid derivatives of Cl-, 3-, y-tocopherols, such as 11-trans-13-octadecatrienoic (or C-eleostearic) acid, trans-9, C-tocopherol hemisuccinate (THS), C-tocopherol Succinate trans-11-trans-13-octadecatrienoic (or (B-eleostearic) acid, and/or mixtures thereof. cis-9, trans-11-cis-13-octadecatrienoic (or punicic) acid, 5.8, 0089. In other some preferred embodiments, endoxifen 11-eicosatrienoic acid, 8,11,14-eicosatrienoic acid and the lipid complexes of the present invention contain sterols. Ste like; tetraenoic unsaturated fatty acids such as 4,8,11,14 rols that find use in the present invention include B-sitosterol, hexadecatetraenoic acid, 6.9,12,15-hexadecatetraenoic acid, Stigmasterol, Stigmastanol, lanosterol, C.-spinasterol, lathos 4,8,12,15-octadecatetraenoic (or moroctic) acid, 6.9,12,15 terol, campesterol and/or mixtures thereof. octadecatetraenoic acid, 9,11,13,15-octadecatetraenoic (or 0090 Compositions of the present invention also include C- or B-parinaric) acid, 9,12,15,18-octadecatetraenoic acid, endoxifen complexes with free and/or salts or esters of fatty 4,8,12,16-eicosatetraenoic acid, 6,10,14, 18-eicosatetraenoic acid. Preferred fatty acids range from those with carbon chain acid, 4,7,10,13-docasatetraenoic acid, 7,10,13,16-docosatet lengths of about C to C, preferably between about C and raenoic acid, 8,12,16,19-docosatetraenoic acid and the like; about Ca, and include tetranoic acid (Co), pentanoic acid penta- and hexa-enoic unsaturated fatty acids Such as 4.8.12. (Cso), hexanoic acid (Co), heptanoic acid (C7o), octanoic 15, 18-eicosapentaenoic (or timnodonic) acid, 4,7,10,13,16 acid (Cso), nonanoic acid (Coo), decanoic acid (Coo), unde docosapentaenoic acid, 4.8,12,15,19-docosapentaenoic (or canoic acid (Clio), dodecanoic acid (Co), tridecanoic acid clupanodonic) acid, 7,10,13,16,19-docosapentaenoic, 4.7. (Co.), tetradecanoic (myristic) acid (Ciao), pentadecanoic 10,13,16,19-docosahexaenoic acid, 4,8,12,15, 18.21-tetra acid (Cso), hexadecanoic (palmatic) acid (Cleo), heptade cosahexaenoic (or nisinic) acid and the like; branched-chain canoic acid (C7o), octadecanoic (stearic) acid (Cso), nona fatty acids such as 3-methylbutanoic (or isovaleric) acid, decanoic acid (Coo), eicosanoic (arachidic) acid (Coo), 8-methyldodecanoic acid, 10-methylundecanoic (or isolau heneicosanoic acid (Co), docosanoic (behenic) acid (C. ric) acid, 11-methyldodecanoic (or isoundecylic) acid, US 2010/01 12041 A1 May 6, 2010
12-methyltridecanoic (or isomyristic) acid, 13-methyltet and thus can be used in drug formulations where the need is to radecanoic (or isopentadecylic) acid, 14-methylpentade have pure forms of these sterones. See, e.g., U.S. Application canoic (or isopalmitic) acid, 15-methylhexadecanoic, 10-me Ser. No. 60/856,952, filed Nov. 6, 2006, and PCT/US07/ thylheptadecanoic acid, 16-methylheptadecanoic (or 83832, filed Nov. 6, 2007, both incorporated herein by refer isostearic) acid, 18-methylnonadecanoic (or isoarachidic) CCC. acid, 20-methylheneicosanoic (or isobehenic) acid, 22-meth 0097. Yet another aspect of the invention provides forming yltricosanoic (or isolignoceric) acid, 24-methylpenta complexes of endoxifen with derivatives of phospholipids cosanoic (or isocerotic) acid, 26-methylheptacosanoic (or Such as pegylated phospholipids. Examples of pegylated lip isomonatonic) acid, 2,4,6-trimethyloctacosanoic (or mycoce ids finding use in the present invention include but are not ranic or mycoserosic) acid, 2-methyl-cis-2-butenoic(angelic) limited to the polyethylene glycol (Pegylated, PEG) deriva acid, 2-methyl-trans-2-butenoic (or tiglic) acid, 4-methyl-3- tives of distearoylphosphatidylglycerol, dimyristoylphos pentenoic (or pyroterebic) acid and the like. phatidylglycerol, dioleoylphosphatidylglycerol and the like. 0091. In some preferred embodiments, endoxifen-lipid 0098. In other aspects, the present invention provides complexes contain phospholipids. Any Suitable phospholip compositions comprising endoxifen and polyethyleneglycol ids or mixture of phospholipids can be used. For example, (PEG) and one or more lipids. phospholipids can be obtained from natural sources or chemi 0099. According to yet other aspects, the present invention cally synthesized. Suitable phospholipids include but are not provides compositions comprising endoxifen complexes limited to phosphatidylethanolamine (PE), phosphatidylg with one or more lipids. Examples include but are not limited lycerol (PG), phosphatidylserine (PS), phosphatidylcholine to compositions comprising endoxifen, cholesterol or choles (PC), phosphatidylinositol (PI), phosphatidic acid (PA), sph terol derivatives and one or more phospholipids. Other ingomyelin and the like, either used separately or in combi examples of compositions include endoxifen, B-sitosterol, nation. Phosphatidylglycerols may be having short chain or and one or more phospholipids. In some preferred embodi long chain, Saturated or unsaturated Such as dimyristoylphos ments, compositions of the present invention comprise phatidylglycerol, dioleoylphosphatidylglycerol, dis endoxifen, and hydrogenated soy phosphatidylcholine or Soy tearoylphosphatidylglycerol, dipalmitoylphosphatidylglyc phosphatidylcholine. erol, diarachidonoylphosphatidylglycerol, short chain phosphatidylglycerol (C-C), and mixtures thereof. 0100. The term “Polyethylene glycol (PEG) includes Examples of phosphatidylcholines includes dimyris polymers of lower alkylene oxide, in particular ethylene toylphophatidylcholine, distearoylphosphatidylcholine, oxide (polyethylene glycols) having an esterifiable hydroxyl group at least at one end of the polymer molecule, as well as dipalmitoylphosphatidylcholine, dioleoylphosphatidylcho derivatives of such polymers having esterifiable carboxy line, diarachidonoylphosphatidylcholine, egg phosphatidyl groups. Polyethylene glycols of an average molecular weight choline, soy phosphatidylcholine or hydrogenated soy phos ranging from 200-20,000 are preferred; those having an aver phatidylcholine can be used, as can mixtures thereof. age molecular weight ranging from 500-2000 are particularly 0092. According to one aspect, the present invention pro preferred. vides compositions comprising endoxifen and derivatives of mono-, di- and tri-glycerides. Examples of the glycerides 0101 Another aspect of the invention provides forming include 1-oleoyl-glycerol (monoolein) and 1,2-dioctanoyl complexes of endoxifen with carbohydrate-based lipids. sn-glycerol. Examples of carbohydrate-based lipids include but are not 0093. Another aspect of the invention provides forming limited to galactolipids, mannolipids, galactolecithin and the complexes of endoxifen with functionalized phospholipids like. including but not limited to phosphatidylethanolamine, pref 0102. In some embodiments of compositions of the inven erably dioleoylphosphatidylethanolamine, phosphatidylthio tion, a complex is formed comprising endoxifen and prefer ethanol, N-biotinylphosphatidylethanolamine and phos ably endoxifen in water at a concentration of about 0.5 phatidylethylene glycol. mg/mL to about 25 mg/mL, such as between 1 mg/mL and 0094. Another aspect of the invention provides forming about 20 mg/mL or between 1 mg/mL and 10 mg/mL, more complexes of endoxifen with carbohydrate-based lipids. preferably between 1 mg/mL and 5 mg/mL. Examples of carbohydrate-based lipids include but are not 0103) In some embodiments, compositions of the present limited to galactolipids, mannolipids, galactolecithin and the invention contain about 2.5% to about 90% of total lipid, like. preferably about 2.5 to about 50% weight of total lipid or 0095. In other preferred embodiment, endoxifen-lipid more, preferably about 10% to about 50% weight of total complexes comprise sterols. Sterols finding use in the present lipid. invention include but are not limited to B-sitosterol, Stigmas 0104. In certain embodiments, compositions of the terol, Stigmastanol, lanosterol, C.-spinasterol, lathosterol, present invention preferably contain endoxifen, and lipid(s) campesterol and/or mixtures thereof. in moleratio between 1:1 to 1:100 such as in between 1:1 and 0096. Another aspect of the invention provides forming 1:20 molar ratio or in between 1:1 and 1:30 molar ratio or in complexes of endoxifen with guggulipid and any Suitable between 1:1 and 1:40 molar ratio or in between 1:1 and 1:50 phospholipids. Guggulipid, or guggul, is a natural Substance molar ratio, in between 1:1 and 1:60 molar ratio, in between derived from the mukul myrrh tree. The mukul myrrh gives 1:1 and 1:70 molar ratios, and in between 1:1 and 1:80 molar off a sticky resin, which is processed to obtain guggulipid. ratios, and 1:90 molar ratios. This extract has been used for thousands of years in 0105 Ratios recited herein, e.g., mole ratios of compo Aryu Vedic medicine to treat arthritis and obesity. The gug nents in a composition, are provided by way of example and gulipid is a source of sterol compounds such as Z- and E-gug do not limit the invention to the precise incremental ratios gulsterones, generally present in an amount of at least 2.5% recited, e.g., to whole number ratios of the components in the (10). Zand E-Guggulsterones can be synthesized chemically composition. For example, a range of ratios of about 1:10 to US 2010/01 12041 A1 May 6, 2010
1:90 encompasses not only 1:11, 1:25, 1:89, etc., but includes, 0114. In other embodiments, the method of the present without limitation, any ratio at or between about 1:10 to 1:90 invention comprises mixing endoxifen, one or more lipids in (e.g., 1:53.637). any suitable order and in any suitable solvents such that the 0106. In certain embodiments, compositions of the resulting composition of the present invention contains present invention preferably contain endoxifen and hydroge endoxifen, and one or more lipids. nated Soy phosphatidylcholine, or soy phosphatidylcholine, 0.115. In some embodiments, the method of preparation of and cholesterol or cholesterol derivative. Such composition the present invention comprises heating the composition includes endoxifen and cholesterol or cholesterol derivative comprising endoxifen, and the lipid(s) at temperatures rang preferably in from about 1:1-1:5 mole ratio, and more pref ing from 30-100° C. preferably between 30-80° C. and more erably at about 1:1 mole ratio to about 1:2 mole ratio. preferably between 30-60° C. 0107 Yet another aspect of the invention is to form com 0116. In some embodiments, the pH of the composition of plexes of endoxifen with derivatives of phospholipids, such as invention ranges from about 3 to about 11, while a pH pegylated phospholipids. Examples include but are not lim between 3.5 to about 8 is preferred and pH of between 4.0 to ited to the polyethylene glycol (PEG) derivatives of dis pH 7.5 are particularly preferred. Aqueous solutions having tearoylphosphatidylglycerol, dimyristoylphosphatidylglyc of a particular pH can be prepared from water having com erol, dioleoylphosphatidylglycerol and the like. prising appropriate buffers. Preferred buffers include but are 0108. In some preferred embodiments, the mole ratio of not limited to mixtures of monobasic sodium phosphate and endoxifen and hydrogenated Soy phosphatidylcholine or Soy dibasic sodium phosphate, tribasic sodium phosphate, diso phosphatidylcholine, in a composition containing endoxifen dium succinate. Other buffers that find use with the present and hydrogenated soy phosphatidylcholine or phosphatidyl invention include Sodium carbonate, sodium bicarbonate, choline is between about 1:10 and 1:90, e.g., between about Sodium hydroxide, ammonium acetate, Sodium citrate, tris 1:10 and 1:80 or 1:10 and 1:70 or 1:10 and 1:60 or 1:10 and (hydroxy-methyl) aminoethane, Sodium benzoate, and the 1:50 or 1:10 and 1:40 and 1:10 and 1:30. Particularly pre like. ferred embodiments, the mole ratio of endoxifen and hydro 0117 The mole ratio of endoxifen and hydrogenated soy genated Soy phosphatidylcholine or soy phosphatidylcholine phosphatidylcholine or soy phosphatidylcholine in the com is between 1:10 and 1:60. position containing endoxifen and hydrogenated soy phos phatidylcholine or soy phosphatidylcholine is in between 0109. In some embodiments, compositions of the present 1:10 and 1:90 Such as in between 1:10 and 1:80 or 1:10 and invention preferably contain endoxifen and total lipids having 1:80 or 1:10 and 1:60 or 1:10 and 1:50 or 1:10 and 1:40 and weight to weight ratio between 1:1 to 1:100 ratio such as 1:10 and 1:30. In preferred embodiments, the mole ratio of between 1:1 and 1:20 ratio or between 1:1 and 1:30 ratio or endoxifen and hydrogenated Soy phosphatidylcholine or Soy between 1:1 and 1:40 ratio or between 1:1 and 1:50 ratio, or phosphatidylcholine is in between 1:5 and 1:60. between 1:1 and 1:60 ratio, or between 1:1 and 1:70 ratio, and 0118. As noted above, compositions can be filtered to between 1:1 and 1:80 ratio, or in between 1:1 and 1:90 ratio. obtain a desired size range of complexes particle sizes from 0110. In some embodiments, the method of the present the filtrate. Filters that find use in the present invention invention comprises solubilizing or Suspending endoxifen include those that can be used to obtain the desired size range and lipid(s) together in an aqueous solution, e.g., water. of the complexes from the filtrate. For example, the com Endoxifen-lipid complex solution can be filtered through plexes can be formed and thereafter filtered through a 5 suitable filters to control the size distribution of the com micron filter to obtain complexes, each particle having a plexes. diameter of about 5 micron or less. Alternatively, 1 um, 500 0111. In some embodiments, the method may comprise nm, 200 nm, 100 nm or other filters can be used to obtain mixing lipid(s) together in water and then adding endoxifen. complexes having diameters of about 1 um, 500 nm, 200 nm, Endoxifen-lipid complex solution can be filtered through 100 nm or any suitable size range, respectively. suitable filters to control the size distribution of the com 0119 When desired, the endoxifen-lipid complex can be plexes. dried, e.g., by evaporation or lyophilization. In certain 0112. In some embodiments, the method also comprises embodiments of the invention, the endoxifen-lipid complex mixing endoxifen and lipid(s) in an organic solvent(s). Such can be lyophilized with one or more cryoprotectants such as as chloroform or ethanol or any other pharmaceutically Sugars. In preferred embodiments, Sugars include but are not acceptable solvents, and evaporating the solvent(s) to form a limited to trehalose, maltose, lactose. Sucrose, glucose, and lipid phase or lipid film. The lipid phase is then hydrated with dextran. In particularly preferred embodiments, trehalose water oran aqueous solution. Examples of aqueous solutions and/or Sucrose are used. Lyophilization is accomplished include but are not limited to 0.9% sodium chloride, solutions under vacuum and can take place either with or without prior containing Sugars such as dextrose, Sucrose, and the like. The freezing of the endoxifen lipid preparation. When desired, the hydrated solution can be filtered through suitable filters to complexes can be resuspended in any desirable solvent control the size distribution of the complexes. including water, Saline, dextrose and buffer. 0113. In some embodiments, the method comprises mix I0120 Pharmaceutical preparations that find use with the ing lipid(s) in an organic solvent(s) and evaporating the Sol compositions of the present invention include but are not vent(s) to form a lipid phase or lipid film. The lipid phase is limited to tablets, capsules, pills, dragees, Suppositories, solu then hydrated with aqueous solution containing endoxifen. tions, Suspensions, emulsions, ointments, and gels. For the The aqueous Solution in addition to endoxifen may further oral mode of administration, preferred forms of endoxifen or contain sodium chloride or Sugars such as dextrose, Sucrose endoxifen lipid complex include tablets, capsules, lozenges, and the like. The hydrated solution can be filtered through powders, syrups, aqueous solutions, Suspensions and the like. suitable filters to control the size distribution of the com For topical application and Suppositories, preferred forms of plexes. endoxifen or endoxifen-lipid complex comprise gels, oils, US 2010/01 12041 A1 May 6, 2010
and emulsions, such as are formed by the addition of suitable I012.8 “Duct ectasia” refers to a dilation of mammary ducts water-soluble or water-insoluble excipients, for example by lipid and cellular debris. Rupture of the ducts induces polyethylene glycols, certain fats, and esters, compounds infiltration by granulocytes and plasma cells. having a higher content of polyunsaturated fatty acids and I0129. “Fibroadenoma’ refers to benign tumors that are derivatives thereof. Derivatives include mono-, di-, and trig derived from glandular epithelium and contain a conspicuous lycerides and their aliphatic esters (for example, fish oils, stroma of proliferating fibroblasts and connective tissue. vegetable oils etc.) or mixtures of these substances. Suitable 0.130 “Fibrosis' simply refers to a prominence of fibrous excipients are those in which the drug complexes are suffi tissue in the breast. ciently stable to allow for therapeutic use. I0131 “Hyperplasia” refers to an overgrowth of cells, 0121 When desired, composition containing endoxifen or where several layers of cells line the basal membrane, without endoxifen-lipid complex can be encapsulated in enteric tumor formation. Hyperplasia increases the bulk of mammary coated capsules to protect it from acids in the stomach. The tissue. In “epithelial hyperplasia, the cells lining breast ducts term “enteric' refers to the small intestine, and enteric coat and lobules are involved, giving rise to the terms “ductal ings prevent release of medication before it reaches the Small hyperplasia” and “lobular hyperplasia. Based on a histologi intestine. Most enteric coatings work by presenting a Surface cal determination, hyperplasia may be characterized as that is stable at acidic pH but breaks down rapidly at higher “usual or "atypical'. pH. I0132) "Metaplasia” refers to a phenomenon in which a 0122 Enteric coating of capsules filled with composition differentiated tissue of one type transforms into a differenti containing endoxifen or endoxifen-lipid complex can be done ated tissue of another type. Metaplasia often results from an environmental change, and enables cells better to withstand as methods known in the art. the change. 0123. The endoxifen-lipid complex of the present inven I0133. The compositions of the present invention may be tion can be of varying size or can be of substantially uniform administered in any dosage form and via any system that size. For example, the complex can have a mean diameter of delivers the active compound endoxifen to breast estrogen about 1 mm or less, and more preferably are in the micron or receptors in vivo. In some embodiments, a composition of Sub-micron range. In some preferred embodiments, the com present invention is delivered by "percutaneous administra plexes have an average diameter of about 5um or less, such as tion', e.g., delivering the drug from the Surface of patient's 0.2 Lum or less or 0.1 um or less. skin, through the stratum corneum, epidermis, and dermis 0.124. The technology outlined in the present invention layers, and into the microcirculations. This is generally may also be used for any other water-insoluble drugs. The accomplished by diffusion down a concentration gradient. methods and compositions of the present invention find use in The diffusion may occur via intracellular penetration conjunction with the methods and compositions disclosed in (through the cells), intercellular penetration (between the U.S. Application Ser. No. 60/850,446, filed Oct. 10, 2006, cells), transappendageal penetration (through the hair fol PCT Application Ser. No. PCT/US07/80984, filed Oct. 10, licles, Sweat, and sebaceous glands), or any combination of 2007, U.S. Application Ser. No. 60/856,952, filed Nov. 6, the above. 2006, PCT Application Ser. No. PCT/US07/83832, filed Nov. 0.134 Percutaneous administration of the endoxifen com 6, 2007, all of which are incorporated by reference herein in position of the present invention may be advantageous their entireties. because this may reduce systemic drug exposure and the risks 0.125. The compositions of the present invention can be from non-specifically activating estrogen receptors through employed to treat breast cancer and breast related diseases. out the body. This is because in topical application of endox For example, the compositions of the present invention may ifen will absorb primarily into local tissues. When the com be administered to a patient diagnosed with benign breast position of invention containing endoxifen will be disease. As used herein, the term “benign breast disease' percutaneously applied to breasts, high concentration will refers to a constellation of non-malignant aberrations in accumulate in the breast tissues presumably due to many breast tissue. The aberrations may be proliferative or non estrogen receptors therein. The composition of endoxifen proliferative in nature. The exemplary benign breast diseases may be applied to any skin Surface, preferably to one or both treatable by the present inventive compositions include breasts. The daily doses to be administered can initially be adenosis, cysts, duct ectasia, fibroadenoma, fibrosis, hyper estimated based upon the absorption coefficients of endox plasia, metaplasia and other fibrocystic changes. Each of ifen, the breast tissue concentration that is desired, and the these diseases, referred as “changes” or “conditions” due to plasma concentration that should not be exceeded. The initial their prevalence, have well-defined histological and clinical dose may be optimized in each patient, depending on indi characteristics. vidual responses. 0126 Adenosis” refers to generalized glandular disease 0.135 Percutaneous administration can be achieved in dif of the breast. It typically involves an enlargement of breast ferent ways, such as (i) by mixing the composition of endox lobules, which contain more glands than usual. In 'sclerosing ifen with Suitable pharmaceutical carriers and, optionally, adenosis,” or “fibrosing adenosis, the enlarged lobules are penetration enhancers to form ointments, emulsions, gel. distorted by scar-like fibrous tissue. lotion, creams or the like, where an amount of said prepara 0127 “Cysts’ are abnormal sacs filled with fluid or semi tion is applied onto a certain area of the skin, (ii) by incorpo solid material. Cysts in the breast are lined by breast epithelial rating the composition of endoxifen into patches or transder cells, developing from lobular structures. They begin as mal delivery systems according to the technology known in excess fluid inside breast glands, but may grow to proportions the art. that stretch Surrounding breast tissue, causing pain. “Fibro 0.136 The effectiveness of percutaneous drug administra cysts’ are cystic lesions circumscribed by, or situated within, tion depends on many factors, such as drug concentration, a conspicuous amount of fibrous connective tissue. Surface area of application, time and duration of application, US 2010/01 12041 A1 May 6, 2010 skin temperature, skin hydration, previous irradiation, physi 0.141. In another preferred embodiment the composition cochemical properties of the drug, and partitioning of the of invention containing endoxifen may also contain one or drug between the formulation and the skin. In some embodi more nonaqueous vehicles, such as alcoholic vehicles. ments, e.g., to enhance percutaneous effectiveness, the com Examples of nonaqueous vehicles include ethyl acetate, etha positions or complexes comprise penetration enhancers that nol, and isopropanol, preferably ethanol and isopropanol. improve percutaneous absorption by reducing the resistance These nonaqueous vehicles may be useful for dissolving both of stratum corneum by reversibly altering its physicochemi the active agent endoxifen and any otherpenetration enhancer cal properties, changing hydration in the stratum corneum, used. They also preferably have a low boiling point, prefer acting as co-solvent, or changing the organization of lipids or ably less than 100° C. at atmospheric pressure, to permit rapid proteins in the intracellular spaces. Such enhancers include evaporation upon contact with skin. In particular, ethanol may but are not limited to organic solvents such as alcohol, effectively contribute to the percutaneous absorption of acetone, dimethylsulfoxide (DMSO), polyethylene glycol, endoxifenby rapidly evaporating upon contact with skin. The propoylene glycol, fatty acids and fatty alcohol and their amount of absolute nonaqueous vehicle in a gel formulation derivatives, hydroxyl acids, pyrrolidones, urea, vegetable ranges from 35% to 99% by weight, preferably between 50% oils, essential oils, and mixture thereof. In addition to chemi to 85% and more preferably between 60% to 75%. cal enhancers, physical methods can increase percutaneous 0142. In another preferred embodiment, the composition absorption. For example, occlusive bandages induce hydra or formulation of the invention comprises an aqueous vehicle tion of the skin. Other physical methods include iontophore that permits solubilization of hydrophilic molecules, and pro sis and Sonophoresis, which use electrical fields and high motes moisturization of skin. An aqueous vehicle also can frequency ultrasound, respectively, to enhance absorption of regulate pH. Aqueous vehicles include alkalinizing and basic drugs that are poorly absorbed due to their size and ionic buffer solutions, including phosphate buffer solutions, characteristics (12-13). Those who are in the pharmaceutical including phosphate buffer Solutions (e.g., dibasic or field can easily manipulate the various factors and methods to monobasic sodium phosphate); citrate buffered solutions achieve right efficacious dosage for percutaneous delivery. (e.g., sodium citrate or potassium citrate) and purified water. 0.137 For percutaneous administration, the formulation or The amount of an aqueous vehicle preferably ranges between composition of the invention containing endoxifen may be 0.1% to 65% by weight of the pharmaceutical composition, delivered in the form of ointment, emulsion (lotion), cream, preferably between 15% to 50%, and more preferably gel, powder, oil or similar formulation. In some embodi between 25% to 40%. ments, the formulation comprises excipient additives, includ 0143. In other embodiments, the composition of the inven ing but not limited to vegetable oils such as Soybean oil, tion comprises one or more gelling agents to increase the mustard oil, almond oil, olive oil, groundnut oil, peanut oil, Viscosity of the composition or formulation or to function as peach kernel oil, groundnut oil, castor oil, canola oil, and the a solubilizing agent. It may constitute between 0.1% to 20% like, animal fats, DMSO, lanolin lipoids, phosphatides, by weight of formulation depending on the nature of gelling hydrocarbons such as paraffin's, petroleum jelly, waxes, leci agent, preferably between 0.5% to 10% and more preferably thin, detergent emulsifying agents, carotin, alcohols, glyc between 0.5% to 5%. The gelling agents may be carbomers, erol, glycerol ether, glycerine, glycol, glycol ethers, polyeth cellulose derivatives, poloxamers and poloxamines. The pre ylene glycol, polypropylene glycol, non-volatile fatty ferred gelling agents are chitosan, dextran, pectins, natural alcohols, acids, esters, Volatile alcoholic compounds, talc, gums and cellulose derivatives such as ethyl cellulose, urea, cellulose derivatives, coloring agents, antioxidants and hydroxyethyl cellulose, hydroxypropyl cellulose, hydrox preservatives. ypropyl methyl cellulose (HPMC), carboxymethyl cellulose 0.138. In some embodiments the formulation or composi (CMC) and the like. The most preferred gelling agent is tion of the invention containing endoxifen may be delivered hydroxypropyl cellulose. as transdermal patch. The patch may comprise (i) a solution 0144. The composition of invention may comprise a gel impermeable backing foil, (ii) a layer like element having a ling agent as described above, in particular a non-preneutral cavity, (iii) a microporus or semipermeable membrane, (iv) a ized acrylic polymer and also comprise a neutralizing agent. self-adhesive layer, and (V) optionally a removable backing The ratio of neutralizing agent/gelling agent varies in film. The layer-like element having a cavity may beformed by between 10:1 to 0.1:1, preferably between 7:1 to 0.5:1, and the backing foil and the membrane. Alternatively, the patch more preferably between 4:1 to 1:1. A neutralizing agent in may comprise (i) a solution-impermeable backing foil. (ii) an the presence of polymer should form salts that are soluble in open-pored foam, a closed pore foam, a tissue like layer or a the vehicle. A neutralizing agent also should permit optimum fibrous web-like layer as reservoir, (iii) a self adhesive layer, Swelling of polymer chains during neutralization of charges and (iv) optionally a removable backing film. and formation of polymer salts. The neutralizing agents 0.139. In some preferred embodiments, the composition of include ammonium hydroxide, potassium hydroxide, Sodium the invention containing endoxifen is formulated in hydro hydroxide, aminomethylpropanol, trolamine, and alcoholic gel and the amount of endoxifen may vary from tromethamine. Those skilled in the art will select a neutraliz 0.001001 to 1.0 gram per 100 grams of gel, most preferably in ing agent according to the type of geling agent used in the the range of 0.01-0.20 grams per 100 grams of gel. composition or formulation. However, no neutralizing agent 0140. In other embodiments, the composition of present is required when a cellulose derivative will be used as geling invention comprises one or more fatty acid esters as a pen agents. etration enhancer. One of the highly preferred examples of a 0145. In some embodiments, the compositions of present fatty acid ester penetration enhancer is isopropyl myristate. invention are employed to treat other diseases, and the medi When isopropyl myristate is used in gel, the amount may cation is selected from a lipophilic or a compound made range e.g., from 0.11 to 5.0 grams per 100 grams of gel. lipophilic by derivatization of the group consisting of antiast preferably from 0.5 to 2.0 grams per 100 grams of gel. hama, antiarrhythmic, antifungals, antihypertensive, antican US 2010/01 12041 A1 May 6, 2010
cer, antibiotics, antidiabetics, antihistamines, antiparasitics, cooled to 0°C. Conc. Sulfuric acid (1 mL) was added drop antivirals, cardiac glycosides, hormones, immunotherapies, wise while maintaining the temperature below room tempera antihypotensives, steroids, sedatives and analgesics, tranquil ture. The solution was stirred at RT for 1 hr. The reaction izers, vaccines, and cell Surface receptor blockers. solution was diluted with hexane and washed with water (1 L) 0146 All references, including publications, patent appli followed by 5% sodium bicarbonate solution (1 L). The cations, and patent cited herein, including those in the list organic layer was dried over Sodium sulfate, filtered and below and otherwise cited in this specification, are hereby evaporated in vacuo at 50–55°C. to give an oil (1.55 Kg). incorporated by reference to the same extent as if each refer Hexane (300 mL) was added to the oil and triturated to give ence was individually and specifically indicated to be incor white solid 3. The suspension was cooled to 0°C. and stirred porated by reference and were set forth in the entirely herein. for 30 min before it was filtered and washed with cold hexane 0147 The use of terms “a” and “an and “the and similar (100 mL) and dried. Yield 1.32 Kg. referents in the context of describing the invention (especially in the context of the following claims) are to be construed to Example 2 cover both the singular and the plural, unless otherwise indi Synthesis of Compound 5 cated herein or clearly contradicted by context. The terms “comprising”, “including”, “having, and "containing are to 0152 be construed as open-ended terms (i.e. meaning “including but not limited to') unless otherwise noted. The use of any and all examples, or exemplary language (e.g., “such as”) Br C provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the speci 1. Mg, O THF fications should be constructed as indicating any non-claimed -- He 2. THF, element as essential to the practice of the invention. Reflux 0148 Preferred embodiments of this invention are O O described, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments can become apparent to those of ordinary O skilled artisans to employ Such variations as appropriate, and the inventors intend for the inventions to be practiced other 3 4 wise than specifically described herein. Accordingly, this C invention includes all modifications and equivalents of the Subject matter recited in the claims appended hereto as per mitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is O encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. OH EXAMPLES 014.9 The following examples further illustrate the inven tion and are not to be construed as in any way as limiting its Scope. O Example 1 O Synthesis of Compound 3
0150 5
Br 0153. Magnesium turnings (115 g) were added to a 10 L Br 4-neck round bottom flask containing anhydrous tetrahydro furan (1 L). The mixture was heated to 55°C. Iodine chips (approx. 5) were added in one lot followed by ethyl bromide H2SO4 -- He (5 mL). Compound 3 (1.1 kg) was dissolved in THF (2L). 200 OO C-RT mL of this solution was added at once to Mg-THF suspension. O O O The reaction was initiated after 30 mins and reflux started. Remaining solution of compound 3 was added drop wise OH 2 maintaining the reflux temperature over a period of 1.5h. The 1 reaction mixture was further refluxed for 2 hr and the cooled to RT. (2-Chloroethoxyphenyl) phenylbutanone (4, 870 g) in 3 THF (1.5 L) was added drop wise over a period of 1 h main taining the temperature between 30-35°C. The reaction mix 0151 4-Bromophenol (1, 1 kg) and 3,4-dihydro-2H-pyran ture was refluxed for 4 h and cooled to RT. The reaction (2, 1.5 L) was mixed together in a round bottom flask and mixture was poured into ice cold 50% hydrochloric acid (3 US 2010/01 12041 A1 May 6, 2010
L). The organic layer was separated and the aqueous layer Example 4 was extracted with THF (3x500 mL). The organic layers were combined, dried over sodium sulfate, filtered and concen Synthesis of Compound I trated to give 5 as oil which was carried over to next step 0156 without further purification. Yield-1.57 kg. Example 3 Synthesis of Compound 6 0154) O O C CH3NH2. le
OH 6 HCHN
O
C OH
0157 To a solution of compound 6 (50 g) in isopropanol (500 mL), monomethyl amine (300 mL) was added and heated for 24 h maintaining the temperature between 70-75° C. The completion of reaction was monitored by TLC (tolu ene:triethylamine, 7:3). The solvent was removed in vacuo. Water (500 mL) was added to the residue and extracted with diisopropyl ether (DIPE, 500 mL). The organic layer was separated and the aqueous layer was back extracted with DIPE (200 mL). The organic layers were combined and washed with water (500 mL), 5% aq. sodium bicarbonate (500 mL), dried over sodium sulfate and filtered. The solvent was removed in vacuo to give a gummy residue. Ethyl acetate (50 mL) was added and heated to dissolve the residue com 0155 Compound (5, 1.57 kg) was dissolved in methanol pletely. The solution was cooled to RT and hexane (50 mL) (6 L) and conc. hydrochloric acid (1.57 kg) was added. The was added and stirred for 12 h. The solid was filtered and solution was refluxed for 5h. Methanol was removed in vacuo washed with cold ethyl acetate-hexane (1:1, 10 mL) mixture. and dichloromethane (5 L) was added. The organic layer was Product I was dried overnight under high vacuum. Yield 25 g. separated. The aqueous layer was extracted with dichlo romethane (2x500 mL). The organic layers were combined Example 5 and washed with water (2 L), 5% aq. NaHCO3 (2 L), water (2 Endoxifen Solution L), dried over sodium sulfate. Charcoal was added and fil tered. The solvent was removed under vacuum to give oil 0158 Endoxifen solution (1 mg/mL) was prepared by (1.38 kg). The oil was triturated with hexane (5 L) with solubilizing endoxifen (10.3 mg) in 0.2% glacial acetic acid vigorous stirring to yield 6 as solid product which was filtered (10 mL). The pH (-5.75) of the solution was adjusted with 1N and dried. Yield 1.07 kg. sodium hydroxide (300 LL). US 2010/01 12041 A1 May 6, 2010
Example 6 The cells were incubated for multiple days (3-7) with Endox ifen (10 nM to 10 uM) and the inhibition of growth were Endoxifen Solution measured by SRB or MTT staining method. The results indi 0159. Endoxifen solution (5 mg/mL) was prepared by cated significant growth inhibition of cells in the presence of solubilizing endoxifen (100mg) in 2% glacial acetic acid (8.6 Endoxifen ranging from 10 to 100%. Endoxifen induce mL). The solution was diluted with 5% dextrose (10.97 mL). growth inhibition or cell killing in different tumor cells indi The pH (-5.56) of the solution was adjusted with 5N sodium cates the usefulness of Endoxifen in the treatment of cancers hydroxide (430 uL). in humans. Example 7 Example 11 Endoxifen Complexes Endoxifen Inhibits Estradiol Dependent Breast Tumor Growth 0160 A suspension of endoxifen, cholesteryl sulfate, and Soy lecithin, is produced by mixing the components together 0164. It is known that Tamoxifen antagonizes estradiol in Water and homogenizing using. e.g., a high pressure dependent breast cancer Xenograft growth (15). Endoxifen homogenizer. The resulting Suspension can be filtered base and endoxifen-citrate in oral dosage form can be simi through 0.2 um filter and then mixed with 7.5% sucrose larly be tested for inhibition of estradiol dependent MCF-7 solution and lyophilized in either vials or in bulk. The particle Xenograft growth. For the animal experiments, female nude size of the resulting complexes is determined using standard mice (Bom: NMRI-nu/nu) per Xenograft experiment, ages 4 procedures, e.g., using a Nicomp particle sizer 380. to 6 weeks and weighing 20 to 24g, are used according to standard protocols. An example of Such a procedure is as Example 8 follows: 0.165 MCF-7 xenografts are developed by passage of Endoxifen Complexes transplantable tumor from a parent tumor established in 0161. A suspension of endoxifen and soy lecithin is pro oophorectomized athymic nude mice treated with estradiol duced by mixing the components together in water and (16-17). homogenizing using, e.g., a high pressure homogenizer. The 0166 Randomly bred female athymic mice are bilaterally resulting Suspension can be filtered through 0.2 um filter and ovriectomized and allowed a 2-week recovery period before then mixed with 7.5% sucrose solution and lyophilized in the implantation of tumor material. The s.c. transplantation of either vials or in bulk. The particle size is determined using the MCF-7 tumor fragments (size, 1x1x1 mm) is done under standard procedures, e.g., using a Nicomp particle sizer 380. anesthesia. The diameter of the tumors is measured regularly, e.g., once weekly, using a caliper-like mechanical instrument Example 9 and the tumor Volume (V) is calculated according to the empirical equation V-(lengthxwidth)/2. The median Vol Toxicity Testing umes of each group are normalized to the initial tumor Vol 0162 Endoxifen was formulated according to Example 6 ume resulting in the relative tumor volume. In all the experi and was tested for toxicity in male Balb/c mice. A single test ments, tumor-bearing mice receive estradiol Supplementation dose at 100 mg/kg or 50 mg/kg was intravenously adminis estradiol valeriate (E2D), 0.5 mg/kg once/wk i.m.. This tered to mice. All the mice died at the 100 mg/kg dose level Supplementation leads to physiologic levels of serum E2 (25 whereas all animals survived at the 50 mg/kg dose level with 984 pg/mL) that are comparable to the human situation (25 no significant loss of body weight. The mice also Survived in 600 pg/mL depending on the follicular phase). the control group with a vehicle control that lacked endox 0.167 Substances: The following substances are used: ifen. Repeat dose toxicity study was conducted with a dose of E2D, Tamoxifen and endoxifen. 25 mg/kg administered consecutively for 3 days with accu 0168 Treatment Modalities: All MCF-7 transplanted ani mulated dose of 75 mg/kg. All the animals in this group mals receive E2D (0.5 mg/kg) injections once a week. After 4 survived. The results are reported in the table below as the weeks, when hormone-supplemented tumors have grown to number of mice Surviving per total. ~0.7-0.8 cm in diameter (180-250 mm), the mice are ran domized into 4 treatment groups of 5-10 mice each. The 5-10 mice are sacrificed as baseline controls for E2D alone. 0169. The treatment groups are: (i) E2D support (0.5 Treatment Dose (mg/kg) Survival.Total mg/kg once/wk i.m.); (ii) E2D Support (0.5 mg/kg once/wk i.m) plus Tamoxifen (0.5 mg-2 mg)/mouse per day, 5 dayS/ Single dose 100 O2 50 44 week by gavage; (iii) E2D Support (0.5 mg/kg once/wk i.m) Repeat dose 25 44 plus endoxifen (0.5 mg-2 mg)/mouse per day, 5 days/week by gavage; (iv) withdrawal of E2D Support. 0170 Suppression of tumor growth in this breast cancer Example 10 tumor model is indicative of therapeutic effect in the treat ment of breast cancer in humans (15). Endoxifen Exhibits Anti-Proliferative Activity Against Different Tumor Cells Example 12 0163 Endoxifen was tested for antiproliferation activity Endoxifen Minimizes Uterotrophic Effect of Estro against various cancer cell lines from Non Small Lung Can gen cer, Breast Cancer, Prostate Cancer, Melanoma Cancer, Ova 0171 It is known that Tamoxifen is a non-steroidal agent rian Cancer, CNS Cancer, Renal Cancer and Colon Cancers. with potent anti-estrogenic effect in animal and in vitro mod US 2010/01 12041 A1 May 6, 2010
els. This pharmacologic property is related to the drugs abil tumor growth reduction indicates the usefulness of IGF-1 as ity to compete with estrogen for estrogen receptors in breast a Surrogate marker for breast cancer. tissues, and to inhibit the stimulatory effect of estrogen on the uterus, vagina and ovaries (18). Example 15 0172 Endoxifen (0.1 mg-2 mg) is administered orally once daily for 28 days to determine the reduction in Endoxifen Prevents Development of Bicalutamide utertrophic effect of estradiol: Female BALB/c mice approxi Induced Gynecomastia and Breast Pain mately 50 days old and weighing 19-20 g are obtained (e.g., 0176 Bicalutamide (CasodexR) is used for treating pros from Charles-River, Inc.) and housed four to five per cage at tate cancer in men. There is growing evidence that IGF-1 may a temperature (23+ 1 C) and light (12 h light/day). The be involved in prostate cancer promotion and progression. It atrophic changes are observed in the mice. There will be three is also known that anti-estrogen agents such as Tamoxifen groups such as vehicle control, Tamoxifen and endoxifen. decrease IGF-1 levels and prevent biculatamide-induced The animals (5-10 mice) are randomly assigned to each gynecomastia in prostate cancer patients (22). Since, endox group. Daily treatments of intact mice with a dose (e.g., 0.1 ifen is an active metabolite of the Tamoxifen anti-estrogen, mg-2 mg) by gavage of Tamoxifen or endoxifen are expected the Silastic slow-release capsules containing endoxifen for to lead to progressive inhibition of uterine and vaginal weight. implant or oral doses of endoxifen (1 mg-10 mg/day) with 0173 Such results will show that endoxifen has better biculatamide are expected to prevent development of bicluta minimizing uterotrophic effect of estrogen than Tamoxifen, mide-induced gynecomastia and breast pain. and that endoxifen finds use as an effective anti-estrogen. Endoxifen blocking of uterine weight gain stimulated by REFERENCES estrogen can also be demonstrated in immature rats. Endox ifen preparations showing the effects described above find (0177 1. Furr BJ, Jordan V C. The pharmacology and use in the treatment of breast cancer as well as otherestrogen clinical uses of Tamoxifen. Pharmacol Ther 1984; 25:127 sensitive conditions. Such as endometriosis, leiomyomata, 205. and benign breast disease, as well as other estrogen-respon 0.178 2. Osborne. Tamoxifen in the treatment of breast sive conditions in men and women. cancer. N Engl J Med 1998: 339:1609-18. 0179. 3. Fisher B, Costantino J. P. Wickerham D L, Red mond CK, kavanah M. Cronin W M, et al. Tamoxifen for Example 13 prevention of breast cancer: Report of the National Surgi cal Adjuvant and Bowel Project P-1 Study. J. Natl Cancer Endoxifen-Caused Decrease of Ki-67 Antigen Insts 1998; 90:1371-88. Expression in Proliferating Breast Cancer Cells 0180. 4. Stearns V. Ullmer L. Lopez J. F. Smith Y. Issacs C, 0.174 Ki-67 is a nuclear non-histone protein. This antigen Hays D. Hot flushes. Lancet 2002: 360:1851-61. is absent in quiescent cells and is expressed in proliferating 0181 5. Otton S V. Ball S E, Cheung SW. Inaba T. cells and is used as a biomarker (19-20). Endoxifen base or Rudolph RL, Sellers E. M. Venlafaxine oxidation in vitro is endoxifen-citrate in oral or injectable form are given to catalyzed by CYP2D6. Br JClin Pharmacol 1996:41:149 Xenograft breast cancer tumor models (e.g., as described 56. above), as well as to breast cancer patients. Immunochemical 0182 7.6. Stearns V. Johnson MD, Rae JM, Morocho A, determination of Ki-67 is done in tumor cells from breast Novielli A, Bhargava P. Hayes DF. Desta Z, Flockhart D.A. cancer tissues from patients, as well as from mice bearing Active Tamoxifen metabolite concentrations after co-ad tumors as described in Example 11. The MIB-1 or similar ministration of Tamoxifen and the selective serotonin antibody available from commercial sources such as DAKO, reuptake inhibitor paroxetine. J Natl Cancer Inst 2003; Carpenteria, Calif. is used for immunochemical localization 95:1758-1764. of antigen. Decrease in Ki-67 antigen expression in animals 0183 8. Y C, Desta Z, Flockhart D A and Skaar T C. and/or breast cancer patients demonstrate the applicability of Endoxifen (4-hydroxy-N-desmethyl-Tamoxifen) has anti endoxifen in treating breast cancers. estrogenic effects in breast cancer cells with potency simi lar to 4-hydroxy-Tamoxifen. Journal of Cancer Chemo therapy and Pharmacology 2005:55:471-478. Example 14 0.184 9. Young Chai Lim, Lang Li, Zeruesenay Desta, Endoxifen Reduces IGF-1 Levels in Breast Cancer Qianqian Zhao, James M. Rae, David A. Flockhart, and Todd C. Skaar. Endoxifen, a Secondary Metabolite of 0175. It is known for humans that Tamoxifen reduces the Tamoxifen, and 4-OH-Tamoxifen Induce Similar Changes levels of circulating insulin-like growth factor I (IGF-1). in Global Gene Expression Patterns in MCF-7 Breast Can IGF-1 has been used as a surrogate biomarker and predicts the cer Cells; JPET, 2006, 318:503–512. effectiveness of Tamoxifen in treatments of breast cancer 0185. 10. Matthew P. Goetz, James M. Rae, Vera J. et al. patients (21). To test the effects of endoxifen preparations of Pharmacogenetics of Tamoxifen Biotransformation Is the present invention, endoxifenbase or endoxifen-citrate are Associated With Clinical Outcomes of Efficacy and Hot given orally or injected to experimental animals bearing lashes. JCO Dec. 20 2005: 9312-93.18. breast cancer tumors. The concentration of IGF-1 levels in 0186 11. Satyavati G. V. Economic and Medical Plant control and Xenografted breast tumor is monitored by estab Research, 5, 47 (1991). lished assays (e.g., ELISA Kit from Diagnostics Systems 0187. 12. Indian Pharmacopea, 1988. Laboratories, London, UK or DAKO, Carpenteria, Calif.). 0188 13. Remington: The Science and Practice of Phar Endoxifen is administered by gavages at 0.5 mg-2 mg per macy (Lippincott Williams & Wilkins (2000), pages 836 mouse per day, 5 days/week. Decrease of IGF-1 levels and 58. US 2010/01 12041 A1 May 6, 2010 16
0189 14. Percutaneous Absorption: Drugs Cosmetics tromethamine, and wherein said neutralizing agent exists at a Mechanisms Methodology edited by Bronaugh and Maibach, neutralizing agent/gelling agent ratio of about 1:1 to about Marcel Becker Publisher (1999). 4:1. (0190. 15. Stephen R. D. Johnston, Irene M. Boedding 44. The composition of claim 40, wherein said hydroalco haus, Sharon Riddler, Ben P. Haynes, Ian R. Hardcastle, holic composition comprises Martin Rowlands, Rachel Grimshaw, Michael Jarman, and (i) endoxifen at about 0.01% to 0.20% by weight; Mitch Dowsett. Iodoxifen Antagonizes Estradiol-depen (ii) isopropyl myristate at about 0.1% to 2.0%, preferably dent MCF-7 Breast Cancer Xenograft Growth through 0.5% to 2.0% by weight; Sustained Induction of Apoptosis, Cancer Research, 59. (iii) alcohol at about 50.0% to 80.0%, preferably about 3646-3651 (1999). 60.0% to 75.0% by weight (0191) 16. Iino, Y. Wolf, DM, Langan-Fahey SM, Johnson, (iv) aqueous vehicle at about 20.0% to 60.0%, preferably DA, Ricchio, M. Thompson, ME, and Jordan V C. Revers 25.0% to 50.0% by weight; and ible Control of Oestradiol-stimulated Growth of MCF-7 (v) gelling agent at about 1.0% to 10.0%, preferably about Tumours in the Athymic Mouse. Br. J. cancer, 64: 1019 0.5% to 5.0% by weight. 1024 (1991) wherein the percentage of components is weight to weight (0192 17. Jann N Sarkaria, David FC Gibson, V C Jordan, of the composition. John F Fowler, Mary J. Lindstrom, and R. Timothy Mulacahy. Tamoxifen-induced increase in the Doubling 45. The composition of claim 40, wherein said endoxifen is time of MCF-7 Xenografts as Determined by Bromodeox in the form of a salt selected from the group of salts consisting yuridine Labeling and Flow Cytometry. Cancer Research of citrate, acetate, formate, oxalate, tartarate, trifluoroacetate, 53, 4413-4417 (1993). methane Sulfonate, phosphate, Sulfate, chloride, bromide, (0193 18. Nanjoo Suh, Andrew L. Glasebrook, Alan D. iodide, lactate, and formate salts. palkowitz, Henry U. Bryant, Lorris L. Burris, james J. 46. The composition of claim 40, wherein said composi tion comprises at least one lipid selected from the group Starling, Homer L. Pearce, Charlotte Williams, Christo consisting of egg phosphatidylcholine (EPC), egg phosphati pher Peer, Yongping Wang and Michael B. Sporn. Arzox dylglycerol (EPG), soy phosphatidylcholine (SPC), hydroge ifene, a New Selective Estrogen receptor Modulator For nated soy phosphatidylcholine (HSPC), dimyristoylphos Chemo prevention of Experimental Breast Cancer. Cancer phatidylcholine (DMPC), dimyristoylphosphatidylglycerol Research 61, 8412-8415 (2001). (DMPG), dipalmitoylphosohatidylcholine (DPPC), dis 0194 19. Assersohn L. Salter J. Powles TJ, et al: Studies teroylphosphatidylglycerol (DSPG), dipalmitoylphosphati of the potential utility of Ki-67 as a predictive molecular dylglycerol (DMPG), cholesterol (Chol), cholesterol sulfate marker of clinical response in primary breast cancer. Breast and its salts (CS), cholesterol hemisuccinate and its salts Cancer Res Treat 82:113-123, (2003). (Chems), cholesterol phosphate and its salts (CP), choles (0195 20. Kenny FS, Willsher PC, Gee JM, et al: Change terylphosphocholine and other hydroxycholesterol or amino in expression of ER, bcl-2 and MIB-1 on primary Tamox cholesterol derivatives, cholesteryl succinate, cholesteryl ole ifen and relation to response in ER positive breast cancer. ate, polyethylene glycol derivatives of cholesterol (choles Breast Cancer Res Treat 65: 135-144, (2001). terol-PEG), coprostanol, cholestanol, cholestane, cholic acid, (0196. 21. Nahta R, HartobagyiGN and Esteva F.J. Growth cortisol, corticosterone, hydrocortisone, and calciferol, factor Receptors in Breast Cancer: Potential for Therapeu E-guggulsterone, Z-guggulsterone, mixture of E- and Z-gug tic Intervention, Oncologist 8:5-17. (2003). gulsterone, monoglycerides, diglycerides, triglycerides, car 0.197 22. Saltstein D, Sieber P. Morris T. Gallo J. Preven bohydrate-based lipids selected from a group consisting of tion and Management of Biclutamide-induced gyneco galactolipid, mannolipid, galactolecithin, B-sitosterol, Stig mastia and Breast Pain: Randomized Endocrinologic and masterol, Stigmastanol, lanosterol, C.-spinasterol, lathosterol, Clinical Studies with Tamoxifen and Anastrozole. Prostate campesterol, phosphatidylcholine, phosphatidylglycerol, cancer and Prostatic Diseases 8, 75-83 (2005). phosphatidylethanolamine, phosphatidylserine, phosphat dylinositol, phosphatidic acid, and pegylated derivatives of 1-39. (canceled) distearoylphosphatidylglycerol, dipalmitoylphosphatidylg 40. A composition comprising an effective amount of a lycerol, dimyristoylphosphatidylglycerol, and dioleoylphos complex comprising endoxifen, wherein said endoxifen is a phatidylglycerol. free base or is in the form of a salt. 47. The composition of claim 40, wherein said composi 41. The composition of claim 40, wherein said composi tion comprises endoxifen, cholesterol and/or a cholesterol tion comprising endoxifen is formulated in a hydroalcoholic derivative, and one or more phospholipids. gel, a hydroalcoholic Solution, a patch, a cream, an emulsion, 48. The composition of claim 47, wherein at least one of a lotion, an ointment, a powder, a tablet, a capsule, an enteric said one or more phospholipids is hydrogenated Soy phos coated capsule, an enteric coated tablet, a lozenge, or an oil. phatidylcholine or soy phosphatidylcholine. 42. The composition of claim 40, wherein said composi 49. The composition of claim 40, wherein said composi tion comprising endoxifen is formulated in a hydroalcoholic tion comprises a form selected from the group consisting of composition containing a penetration enhancer, an aqueous powder, Solution, emulsion, micelle, liposome, lipidic par vehicle, an alcoholic vehicle and a gelling agent. ticle, gel, paste form, a lyophilized form, and a tablet or a 43. The composition of claim 40, wherein said hydroalco filled capsule, wherein said tablet or filled capsule optionally holic composition comprises a neutralizing agent, wherein comprises an enteric coating material. said neutralizing agent is selected from the group consisting 50. The composition of claim 40, wherein said composi of Sodium hydroxide, potassium hydroxide, ammonium tion is in a lyophilized form comprising a cryoprotectant, hydroxide, aminomethylpropanol, arginine, trolamine, and wherein said cryoprotectant comprises one or more Sugars US 2010/01 12041 A1 May 6, 2010
selected from the group consisting of trehalose, maltose, lac 53. The method of claim 52, further comprising a step of tose, Sucrose, glucose, and dextran. preparing said compound of Formula 3 by reacting com 51. A method of preparing endoxifen, comprising: pound of Formula 1 i. reacting a compound of Formula 5 with acid, wherein the compound of formula 5 has the structure:
Br C
OH OH with a compound of Formula 2. O O
and 54. The method of claim 51, comprising: ii. after said reaction of said compound of Formula 5 with a) crystallizing said endoxifen; and/or acid, reacting said compound with methylamine. b) chromatographically treating said endoxifen; 52. The method of claim 51, comprising a step of preparing said compound of Formula 5 by reacting compound of for to produce a purified preparation of endoxifen, wherein mula 4 4 said purified preparation of endoxifen contains pre dominantly E-isomer, predominantly Z-isomer, or a mixture of E- and Z-isomers of endoxifen. 55. A method of treating a cell, comprising, exposing said cell to a composition of claim 40. 56. The method of claim 55, wherein the cell is in vivo in a host. 57. The method of claim 56, wherein said host is a mam mal. 58. The method of claim 55, wherein said exposing com ac prises administering a composition of claim 40 to a Subject, with a compound of Formula 3. wherein said administering comprises oral, intravenous, Sub cutaneous, percutaneous, parenteral, intraperitoneal, rectal, Br vaginal, and/or topical delivery said composition to said Sub ject. 59. A method of treating a Subject having cancer, compris ing administering a composition of claim 40 to said Subject, wherein said administering comprises oral, intravenous, Sub O O cutaneous, percutaneous, parenteral, intraperitoneal, rectal, vaginal, and/or topical delivery said composition to said Subject.